Project Summary/Abstract DNA damage is the major cause of genomic instability, a hallmark of cancer. DNA-protein crosslinks (DPCs) are one of most toxic forms of DNA damage, interfering with the DNA replication and transcription machineries due to their bulky nature. However, despite the importance of repairing DPCs, very little is known about the DPC repair process. Recent studies, including studies from our lab, identified Spartan (SPRTN, also known as DVC1) as a nuclear metalloprotease that associates with PCNA and plays a critical role in DPC repair at DNA replication forks. Our published studies demonstrated that SPRTN hypomorphic mice exhibited genomic instability, progeria, and liver cancer: all of the major phenotypes in human Ruijs-Aalfs Syndrome, which is caused by inactivating mutations in SPRTN. These results underscore the significance SPRTN in DPC repair and the maintenance of genomic stability. However, it is not known whether other proteases are also involved in DPC repair. Here, we describe our preliminary studies of a novel human protease, which we identified using bioinformatics analyses to search for additional human proteins with domain features similar to SPRTN. Like SPRTN, this protease contains a putative protease domain (trypsin-like peptidase) and a PCNA-interacting peptide (PIP) box. Our preliminary experiments showed that depletion of the protease resulted in accumulation of DPCs, leading us to hypothesize that it also participates in DPC repair. Moreover, low levels of expression of the protease in neuroblastoma are associated with increased genomic instability, suggesting that its deficiency causes genomic instability and cancer. To test these hypotheses, we propose to: 1) study the relationship between the novel protease and SPRTN in DPC repair and sensitivity to DPC-inducing chemotherapeutics; 2) define the functional domains required for DPC repair; and 3) examine genome stability and cancer susceptibility in a knockout mouse model. This project will not only elucidate the mechanism of protease- dependent DPC repair, but will also provide new insight into genome instability and tumorigenesis caused by DPCs, and possibly identify new strategies for cancer therapy.